Loss of the Nuclear Protein RTF2 Enhances Influenza Virus Replication

FIG 1. Screen identifies RTF2 as a potential antiviral candidate gene. (A) Schematic overview of the CRISPR screen for sgRNAs that permit infection in the presence of type I interferon. (B) Volcano plot showing enrichment of sgRNAs in infected cells versus uninfected cells based on the fold change in expression (x axis) and statistical significance (y axis). Blue text shows known genes of the IFN pathway. (C) IAV infection rates based on HA surface protein levels in A549 cells transduced with single sgRNAs and Cas9 lentiviruses (in triplicate). Cells were selected with puromycin for 7 days, pretreated with 200-U/ml IFN-β for 24 h, and infected with IAV at an MOI of 5 (adjusted for cell counts) for 16 h. P values were determined by one-way ANOVA and Dunnett’s multiple-comparison test against nontargeting sgRNA 1. ****, P ≤ 0.0001; ***, P ≤ 0.001; ns, not significant. (D) Schematic showing the distribution of the different RTF2 sgRNAs (boxed numbers) along the consensus coding sequence of RTF2. (E) IAV infection rates based on HA surface protein levels in A549 cells transduced with Cas9 and individual RTF2 sgRNAs. P values were determined by one-way ANOVA and Dunnett’s multiple-comparison…

FIG 2. The loss of RTF2 increases the IAV infection rate in interferon-treated cells. (A) IAV infection rates based on HA surface protein levels in RTF2-KO cells and RTF2-rescued cells. RTF2-KO cells were transduced with either an empty vector or an sgRNA-resistant RTF2 cDNA to restore RTF2 expression. ****, P ≤ 0.0001, by one-way ANOVA and Tukey’s multiple-comparison test. (B) (Top) IAV infection rates based on HA surface protein levels in A549 cells transduced with nontargeting sgRNA, IFITM3 sgRNA, or RTF2 sgRNA. The three rightmost columns denote three separate RTF2-KO cell clones isolated and cultured from a pool of RTF2 sgRNA 7 polyclonal cells. P values were determined by one-way ANOVA and Dunnett’s multiple-comparison test against the nontargeting sgRNA sample. ****, P ≤ 0.0001; ***, P ≤ 0.001. (Bottom) The Western blot (immunoblot [IB]) image shows RTF2 protein expression in the isolated RTF2-KO cell clones. (C) Quantification of IAV progeny virions produced by infected WT A549, RTF2-KO, and RTF2-rescued cells. Cells were first pretreated with 200-U/ml IFN-β for 24 h, before IAV infection at an MOI of 5 for 16 h. The cell culture supernatant was collected and used to perform…

FIG 3. RTF2 localizes to the nucleus. (A) Immunofluorescence of WT A549 cells and two distinct clones of RTF2-KO cells. (B) Biochemical fractionation of WT A549 cells into cytosolic and nuclear fractions. Cells were first suspended in HEPES-sucrose-Ficoll (HSF) buffer containing digitonin to extract the cytosolic proteins and washed once with HSF buffer, before lysing the nuclear pellet in RIPA buffer to release the nuclear proteins. TATA-binding protein (TBP), a nuclear protein, and tubulin, a cytosolic protein, were included as controls for the fractionation protocol. (C) Live-cell imaging of cells expressing either mCherry or the RTF2-mCherry fusion protein. WT A549 (top) and RTF2-KO cells (bottom) were transduced with lentiviruses that encode either FLAG-mCherry or RTF2-FLAG-mCherry and cultured for a week, before they were visualized under a wide-field epifluorescence microscope.

FIG 4. Mislocalization of RTF2 reduces its protective role against IAV. (A) Clonal RTF2-KO cells were transduced with lentiviruses that encode the empty vector, the guide-resistant form of RTF2, or RTF2 with the predicted NLSs mutated. Site-directed mutagenesis was performed to replace lysine or arginine residues with alanine residues. Shown are transduced cells stained with DAPI and anti-RTF2 antibody. RTF2 with intact NLSs localizes to the nucleus. Editing the predicted monopartite NLS had no effect on RTF2’s nuclear localization. However, editing the predicted bipartite NLS seemed to disperse RTF2 throughout the cell. (B) Western blot of WT A549 cells and RTF2-KO cells rescued with either the empty vector or various sgRNA-resistant forms of RTF2: RTF2 (WT RTF2 with intact NLSs), N-terminus-myristoylated (N-myr) RTF2 (RTF2 containing point mutations in both NLSs with an additional plasma membrane-targeting myristoylation signal added to its N terminus), an NLS mutant (RTF2 containing point mutations in both NLSs), or an ER-retention mutant (RTF2 containing point mutations in both NLSs with an additional signal peptide and ER-retaining KDEL motif added). (C) Immunofluorescence of WT…

FIG 5. RTF2 restricts a nuclear stage of the influenza virus life cycle and at least blocks primary transcription. (A) IAV polymerase activity was measured via a minigenome luciferase reporter assay. Cells were transfected with plasmids harboring the genes for IAV polymerase subunits and NP, a reverse-orientation firefly luciferase reporter on a vRNA backbone, and a Renilla luciferase as a transfection control. Luciferase activity was measured at 24 h posttransfection. ****, P ≤ 0.0001, by one-way ANOVA and multiple comparisons. (B) Effect of mislocalizing RTF2 on IAV replication/transcription, as assayed in a minigenome luciferase reporter assay. The cells described in the legend to Fig. 4B were transfected with the plasmids described in panel A. Luciferase activity was measured at 24 h posttransfection. **, P ≤ 0.01, by one-way ANOVA and Dunnett’s multiple-comparison test. (C) The transcript levels of transfected plasmids harboring the genes for IAV NP, PA, PB1, and PB2 were quantified in WT A549, RTF2-KO, and RTF2-rescued cells. Cells were transfected for the minigenome luciferase reporter assay as described in the legend to panel B, and RNA was extracted at 24 h posttransfection for…

FIG 6. RTF2 may affect the upregulation of antiviral ISGs in response to IFN exposure. (A) Overexpressing RTF2 does not confer additional protection against IAV infection in WT A549 cells. WT A549 cells were transduced with either the empty vector or RTF2. After 8 days of blasticidin selection posttransduction, cells were treated with 200-U/ml human IFN-β for 24 h before IAV infection at an MOI of 5 for 16 h and sorted by FACS based on HA. ****, P ≤ 0.0001, by two-way ANOVA and Dunnett’s multiple-comparison test. (B to D) IFN pretreatment enhances the differences in infection rates, as measured by HA staining, between RTF2-KO cells and WT A549/RTF2-rescued cells. (B) Cells pretreated with IFN-β or mock treated. (C) Cells pretreated with IFN-β with or without 0.5-μg/ml B18R, an IFNAR decoy protein. (D) Cells pretreated with IFN-β with or without 5 μM ruxolitinib, a JAK inhibitor. Shown are combined data from two independent experiments with three technical replicates each. P values were determined by one-way ANOVA followed by an unpaired t test with Bonferroni’s correction between RTF2-KO and RTF2-rescued cells. ****, P ≤ 0.0001; **, P ≤ 0.01. (E) The RTF2 mRNA level (normalized to the…

FIG 7. RTF2-KO cells have higher viral RNA levels when infected with other IAV strains and WT VSV. (A, B) WT A549, RTF2-KO, and RTF2-rescued cells were pretreated with 200-U/ml IFN-β before infection with the influenza virus A/New Caledonia/20/1999 (A) or A/California/04/2009 (B) at an MOI of 5 for 5 h. RNA was harvested at 5 hpi for qPCR to measure NP mRNA levels. (C) WT A549, RTF2-KO, and RTF2-rescued cells were pretreated with 200-U/ml IFN-β before infection with WT VSV at an MOI of 5 for 24 h, before RNA was harvested for qPCR to measure N mRNA levels. P values were determined by one-way ANOVA and Tukey’s multiple-comparison test. ****, P ≤ 0.0001; **, P ≤ 0.01; *, P ≤ 0.05.